One-Pot Chemoenzymatic Cascade for the Enantioselective C(1)-Allylation of Tetrahydroisoquinolines

Herein, we report a one-pot, chemoenzymatic process for the synthesis of enantioenriched C(1)-allylated tetrahydroisoquinolines. This transformation couples a monoamine oxidase (MAO-N)-catalyzed oxidation with a metal catalyzed allylboration, followed by a biocatalytic deracemization to afford allylic amine derivatives in both high yields and good to high enantiomeric excess. The cascade is operationally simple, with all components added at the start of the reaction and can be used to generate key building blocks for further elaboration.

MHz for 13 C) in CDCl 3 using residual protic solvent as an internal standard. Reported chemical shifts (δ) (in parts per million (ppm)) are relative to the residual protic solvent signal (CHCl 3 in CDCl 3 , 1 H = 7.26; 13 C = 77.0). Chiral HPLC was performed on an Agilent system (Santa Clara, CA, USA) equipped with a G1379A degasser, G1312A binary pump, a G1367A well plate autosampler unit, a G1316A temperature controlled column compartment and a G1315C diode array detector. CHIRALPAK®IA, CHIRALPAK®IC and CHIRALPAK®IE Analytical (all Daicel (Osaka, Japan), 250 mm length, 4.6 mm diameter, 5 μm particle size) as well as CHIRALCEL®OD-H Analytical (Daicel (Osaka, Japan), 250 mm length, 4.6 mm diameter, 5 μm particle size) columns were used. The typical injection volume was 10 μl and chromatograms were monitored at 265 nm, unless A single colony was used to inoculate a pre-culture (6 mL) (containing LB medium and ampicillin 100 mg/mL) which was grown at 37 °C and 200 rpm for 12 hours (OD 600 between 0.6-1.0). 2 L Erlenmeyer flasks containing 600 mL auto-induction media and ampicillin (100 mg/mL) were inoculated with 6 mL of pre-culture and incubated following Table 1. The cells were harvested by centrifugation at 4000 rpm at 4 °C for 30 minutes. The pelleted cells were stored at -20 °C before using.

General procedure for the purification of monoamine oxidase biocatalysts
5 g of frozen cell pellet was defrosted on ice and resuspended in 25 mL of buffer A (100 mM KPi, pH 7.8, 300 mM NaCl, 30 mM imidazole) containing lysozyme from chicken egg white (1 mg/mL) and incubated at 30 °C for 30 minutes. The suspension was cooled on ice and the cells lysed by ultra-sonocation (20 s on, 20 s off: 20 cycles). The cells were concentrated by centrifugation (18000 rpm, 45 minutes). Subsequently, the cell free extracts were filtered through a syringe with 0.4 μm followed by 0.22 μm pore sizes. The filtered cell-free extracts were loaded onto a HisTrap Ni-sepharose column (1 mL, GE Healthcare) pre-equilibrated with buffer A (100 mM KPi pH 7.8, 300 mM NaCl, 30 mM imidazole). The loaded column was washed with 5 CV (column volumes) of buffer A before eluting with buffer B (100 mM KPi pH 7.8, 300 mM NaCl, 500 mM imidazole) and collecting 1 mL fractions. The enzyme containing fractions (measured using NanoDrop™ spectrophotometer at 280 nm) were pooled before concentrating with Vivaspin® (20 mL, 30 kDa MCO, GE Healthcare) and desalted by eluting with 100 mM KPi pH 7.8. The purified enzymes were snap-frozen in liquid nitrogen and stored at -80 °C before use.

General procedure for the recombinant expression of imine reductase biocatalysts
IRED variants were transformed into BL21 (DE3) competent E. coli (NEB) following manufacturer's instructions. A single colony was used to inoculate a pre-culture (20 mL) (containing LB medium and kanamycin 50 mg/mL), which was grown at 37 °C and 200 rpm for 12 hours (OD 600 between 0.6-1.0). 2 L Erlenmeyer flasks containing 400 mL auto-induction media, kanamycin (50 mg/mL) and 1.6 mL glycerol were inoculated with 20 mL of pre-culture and incubated at 37 °C untill reaching an OD 600 of 0.6. After this time IPTG (0.1 mmol) was added and the flasks left to grow at 25 °C overnight. After this time, the cells were harvested by centrifugation at 4000 rpm at 4 °C for 30 minutes. The pelleted cells were stored at -20 °C before using.

General procedure for the preparation of enzyme lysate
5 g of frozen cell pellet was defrosted on ice and resuspended in 25 mL of phosphate buffer (100 mM KPi pH 7.8) containing lysozyme from chicken egg white (1 mg/mL) and incubated at 30 °C for 30 minutes. The suspension was cooled on ice and the cells lysed by ultra-sonocation (20 s on, 20 s off: 20 cycles). The cells were concentrated by centrifugation (18000 rpm, 45 minutes). Subsequently, 20 mL of cell-free extract was transferred to a 50 mL falcon tube. The falcon tube was snap frozen in liquid nitrogen prior to freezedrying for 1-2 days (until powdery). The freeze-dried lysate was stored at -20 °C prior to use.

Chemical oxidation of substituted tetrahydroisoquinolines
General procedure: To a solution of 1,2,3,4-tetrahydroisoquinoline (1.0 equiv.) in anhydrous DCM at r.t under nitrogen was added N-bromosuccinimide (NBS) (3.0 equiv.). The reaction mixture was stirred at r.t for 1 hour. Afterwards, 30% v/v NaOH aqueous solution was added and left to react for a further 1 hour at r.t. After which time, the aqueous phase was extracted with DCM (3 x 5 mL). The combined organic layers were dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.

Preparation of allylic Grignards
Mg turnings (1.5 equiv.) were stirred in a two-neck round-bottom flask fitted with a condenser, under N 2 for 15 minutes before addition of dry Et 2 O (1.1 mL mmmol -1 ) and a single crystal of I 2 . Subsequently, allylic bromides (2.0 equiv.) were added dropwise and the reaction initiated with a heat gun. The reaction mixture was left to stir for 30 minutes before allowing to cool and being taken through to the next step without further purification or characterisation.

Preparation of 1-substituted tetrahydroisoquinolines
The corresponding allylic Grignard (2.0 equiv.) was added dropwise to a cooled (-25 °C) solution of 3,4-dihydroisoquinoline (1.0 equiv.) and BF 3 .OEt 2 (1.1 equiv.) in dry THF. The reaction was stirred at -25 °C for 2 hours before being allowed to warm to r.t and left to react overnight. After this time, the reaction was quenched by addition of water and left to stir for a further 1 hour. Subsequently, the mixture was extracted with EtOAc (3 x 5 mL), dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.

4,4,5,5-Tetramethyl-2-(2-methylenebutyl)-1,3,2-dioxaborolane, 4b
2-Ethylacrolein (2.33 mL, 23.8 mmol, 1.0 equiv.) was dissolved in a 1:8 mixture of MeOH (2 mL) and Et 2 O (16 mL) before being cooled to 0 °C. Sodium borohydride (0.88 g, 23.8 mmol, 1.0 equiv.) was added portion-wise. The resulting mixture was stirred at 0 °C for 1 hour before being diluted with Et 2 O (20 mL), washed with water (20 mL). The organics were dried (MgSO 4 ) and concentrated in vacuo to give the crude product, which was taken to the next step with no further purification. PBr 3 (3.70 g, 17.8 mmol, 0.75 equiv.) was added dropwise to a solution of the crude alcohol from the above reaction (23.8 mmol, 1.0 equiv.) in dry Et 2 O (20 mL) at 0 °C under an atmosphere of nitrogen. Once the addition was complete the reaction mixture was allowed to warm to room temperature and was left to stir for 12 hours. The reaction mixture was cooled to 0 °C before being quenched by the addition of iced water (20 mL). The organic layer was washed successively with water (20 mL), NaHCO 3 (20 mL) and brine (20 mL). Due to the volatility of the compound the reaction was concentrated to ¼ of the volume and used in the next step as a solution in Et 2 O.
Anhydrous THF (40 mL) was added to a multi-neck flask containing heat activated magnesium turnings (347 mg, 14.3 mmol, 1.2 equiv.), followed by HBPin (1.5 mL, 11.89 mmol, 1.0 equiv.). Half the crude bromide solution prepared as described above was added to the mixture dropwise at room temperature under an atmosphere of nitrogen. After stirring for 30 minutes, the remainder of the bromide solution was added dropwise to the reaction mixture. After stirring at room temperature for 12 hours, the reaction mixture was diluted with hexane and quenched by the slow addition of HCl (40 mL, 0.1 M). The crude compound was extracted into hexanes, dried (MgSO 4 ) and concentrated in vacuo. NMR and GCMS data showed the reaction to be pure and therefore no purification required to give the desired compound as a pale yellow oil (822 mg, 4.19 mmol, 18% (over 3 steps)).

Solvent Screening for the chemical allylation of 3,4-dihydroisoquinoline
To a 2 mL Eppendorf tube was added 5 mM of 3,4-dihydroisoquinoline (0.5 M in DMSO) along with 20 mM Allyl BPin (1 M in DMSO) (See Table 1). Solvent was added to give a 500 µL total reaction volume. Biotransformations were incubated at 30 °C for 24 h with 200 rpm shaking. Reactions were quenched by the addition of 40 μL 10M NaOH followed by 500 μL of MTBE before centrifugation at 13,200 rpm for 1.5 minutes. The organic layer was extracted, dried over anhydrous MgSO 4 before further centrifugation at 13,200 rpm for 1.5 minutes. The organic product was transferred to a GC vial for analysis by GC/chiral HPLC.

Analytical scale procedure for the chemoenzymatic allylation of tetrahydroisoquinoline using purified enzyme
To

Analytical scale procedure for the chemoenzymatic allylboration of tetrahydroisoquinoline using purified enzyme biocatalysts and L.A metal catalyst
To a 2 mL Eppendorf tube was added 5 mM of 1,2,3,4-tetrahydroisoquinoline (1 M in DMSO) along with 40 mM Allyl BPin (1 M in DMSO) and 10 mol% Lewis Acid catalysts (100 mM in dH 2 O). 2 mg mL -1 of purified MAO-N was added prior to addition of 100 mM KPi pH 7.8 to give a 500 µL total reaction volume. Biotransformations were incubated at 30 °C for 24 h with 200 rpm shaking. Reactions were quenched by the addition of 40 μL 10M NaOH followed by 500 μL of MTBE before centrifugation at 13,200 rpm for 1.5 minutes. The upper organic layer was extracted, dried over anhydrous MgSO 4 before further centrifugation at 13,200 rpm for 1.5 minutes. The organic product was transferred to a GC vial for analysis by GC/chiral HPLC.

Analytical scale procedure for the enzymatic deracemisation of (rac)-3a using non-selective chemical reducing agents
To a 2 mL Eppendorf tube was added 5 mM of racemic amine substrate, 3a (1 M in DMSO) along with 50 mM non-selective reducing agent (1 M in DMSO) and 2 mgmL -1 purified MAO-N. Finally, 100 mM KPi pH 7.8 was added to give a 500 µL total reaction volume. Biotransformations were incubated at 30 °C for 24 h with 200 rpm shaking. Reactions were quenched by the addition of 40 μL 10 M NaOH followed by 500 μL of MTBE before centrifugation at 13,200 rpm for 1.5 minutes. The upper organic layer was extracted, dried over anhydrous MgSO 4 before further centrifugation at 13,200 rpm for 1.5 minutes. The organic product was transferred to a GC vial for analysis by GC/chiral HPLC.

Analytical scale procedure for the screening of IREDs for the deracemisation of (rac)-3a
To a 2 mL Eppendorf tube was added 5 mM of racemic amine substrate, 3a (

Absolute configuration
The absolute configurations of optically active products were assigned by comparison with previous reports of the stereoselectivity of the amine oxidase and the imine reductase. All previously reported examples for the oxidation of racemic amines with MAO-N D11 display (S)-selective oxidation. 15-17 R-IRED is an (R)-selective reductase, and many examples have been reported of the Rselective reduction of 3,4-dihydroisoquinoline substrates. When coupling these two enzymes in a deracemisation process, a high degree of stereoselectivity will only be observed when the selectivity of both enzymes is complementary, generating (R)-configured products. The absolute configuration of (R)-3a was robustly assigned by optical rotation (and compared to literature), this result was then used to assign the absolute stereochemistry of the other substrates.

Chiral HPLC and GC.MS data for the MAO-N D11 catalysed kinetic resolution of (rac)-3a
The left figures show the GC and corresponding MS trace for the kinetic resolution of (rac)-3a with MAO-N D11. In the GC.MS data, no imine intermediate is observed. The right figure shows the chiral HPLC trace for the kinetic resolution of (rac)-3a with MAO-N D11. In the chiral HPLC trace, both enantiomers of (rac)-3a can be seen, showing slight preference for the R-enantiomer through the resolution. The right most peak in the chiral HPLC has been tentatively assigned as the allylic imine intermediate (although we were unsuccessful in chemically synthesising this species). If the area% from the HPLC of the minor (S)-enantiomer is summed with the area% of the imine intermediate, this equals the area% of the major (R)-enantiomer, which provides good evidence that this is a kinetic resolution process.  1-Allyl-6-chloro-1,2,3,4-tetrahydroisoquinoline, (R)-3c The upper figures show the GC and corresponding MS trace for the imine intermediate 2c and the racemic product standard (rac)-3c, along with the chiral HPLC chromatogram for (rac)-3c. Below is outlined the GC.MS and corresponding chiral HPLC chromatogram for the enantioselective chemoenzymatic allylation of 1c with allyl boronic acid pinacol ester.  1-Allyl-6,7-dimethoxy-1,2,3,4

-tetrahydroisoquinoline, (R)-3g
The upper figures show the GC and corresponding MS trace for the imine intermediate 2g and the racemic product standard (rac)-3g, along with the chiral HPLC chromatogram for (rac)-3g. Below is outlined the GC.MS and corresponding chiral HPLC chromatogram for the enantioselective chemoenzymatic allylation of 1g with allyl boronic acid pinacol ester.

S41
The upper scheme outlines the diastereoselectivity which is observed during the chemical addition of cis-crotyl BPin to 3,4dihydroisoquinoline. This diastereoselectivity has been previously outlined in literature, and results from the orientation of the methyl group of the BPin in the favoured chair transition state during the reaction. 18 The chiral HPLC traces clearly shows the formation of only two diastereomers, which can be compared to the HPLC trace of (rac)-3s showing all four diastereomers. The scheme below shows the chemoenzymatic cascade for 1,2,3,4-tetrahydroisoquinoline and cis-crotyl BPin. As described above, the cascade initially generates the (R,S)-and (S,R)-diastereomers. The next step involves the selective oxidation of the (S,R)-diastereomer to the corresponding imine, catalysed by MAO-N D11. The chiral imine which is generated then undergoes in situ epimerization, a process which has been observed previously in our group. 19 The IRED is then able to preferentially reduce a single enantiomer of the chiral imine, leading to the accumulation of the (R,S)-product.

(R)-1-(2-Methylbut-3-en-2-yl)-1,2,3,4-tetrahydroisoquinoline, 3q
The  Finally, 10 mg mL -1 of R-IRED cfe was added and the volume made up to 40 mL with 100 mM KPi pH 7.8. Biotransformations were incubated at 30 °C for 48 h with 200 rpm shaking. The reaction was quenched by the addition of 0.5 mL 10M NaOH followed by centrifugation at 4000 rpm for 5 minutes, this was repeated a further two times. The aqueous components were collected and extracted with MTBE (3 x 40 mL). The combined organic layers were dried over anhydrous magnesium sulphate, filtered, and concentrated in vacuo to furnish the desired product (61 mg, 0.60 mmol, 59%) as a dark orange oil. 1

Time course Experiments
To improve our understanding of the reaction, we carried out a series of time course experiments, where we monitored the conversion and enantioselectivity of the formation of 1-allyl-1,2,3,4-tetrahydroisoquinoline 3a from 1,2,3,4-tetrahydroisoquinoline 1a over time (Figure 2a).
After only 6h the conversion reached 90% and 90% ee, and after 12 hours only the desired product and enantiomer could be observed (>99% ee). Interestingly, during these reactions no imine 2a was observed, therefore we ran our time course monitoring the formation of the imine intermediate 3,4-dihydroisoquinoline 2a (Figure 2b) for comparison. This suggests that during our MAO-N/IRED cascade conditions, as soon as intermediate 2a is formed it reacts with the boryl reagent to form the racemic product, alternatively it is reduced back to the starting material by R-IRED to begin the cycle again.